ALBERT

Description: Background: Platelets (PLTs) are currently stored at 22°C (RT, room temperature) for clinical purposes. This approach ensures long circulation time but has numerous downsides, including limited storage time due to the risk of bacterial growth and increased costs due to bacterial testing or pathogen reduction processing. PLTs stored at 4°C were the standard of care in the 1960s and 1970s. In our previous study with healthy volunteers, we showed that humans who received cold-stored PLTs have a significantly weaker response to collagen (an agonist that acts predominantly via GPVI) compared to RT-stored PLTs. If and how cold-stored PLTs recover their function in vivo is poorly understood. Methods: We obtained human PLTs by an apheresis collection and sampled either at baseline (fresh) or after five days at RT or 4°C. To test the response to GPVI-dependent agonists, we stimulated platelet-rich plasma or washed PLTs with collagen and the GPVI-specific agonist convulxin (CVX) and tested for activated integrin and α-degranulation by flow cytometry. Platelet aggregation, in response to GPVI-dependent agonists, was tested by aggregometry. We checked for GPVI expression levels by flow cytometry and for signaling events downstream of GPVI by immunoblotting. To allow for recovery of function in vitro, we incubated either 4°C-stored, or RT-stored PLTs with fresh, platelet-depleted blood for 15min, and perfused the reconstituted whole blood through a microfluidic block and post device to quantify the contractile forces of platelet aggregates. Additionally, we performed platelet force measurements at the single cell level using a traction force microscopy approach. To validate a murine model of platelet storage and transfusion, we replicated functional studies in vitro by testing mouse PLTs for integrin activation and α-degranulation by flow cytometry. Platelet aggregation in response to collagen, CVX, and the GPVI-specific antibody JAQ-1 with crosslinking anti-IgG was also tested. To evaluate the platelet function after transfusion, we obtained whole blood from UbiC-GFP mice and isolated platelet-rich plasma followed by storage for 24 hours at either 4°C or RT. To allow tracking of stored PLTs in vivo, we transfused the UbiC-GFP PLTs into wild-type C57BL/6J mice and tested for integrin activation of endogenous and transfused PLTs. Results: In human PLTs, we found a significantly increased integrin response in 4°C-stored PLTs stimulated with collagen in flow cytometry studies in vitro. Similarly, the aggregation response of 4°C-stored PLTs to collagen was significantly increased compared to RT-stored PLTs in vitro. In line with these findings, we observed more PLCγ2 phosphorylation and Syk phosphorylation at baseline in 4°C-stored PLTs compared to RT-stored PLTs, suggesting more pre-activation downstream of GPVI. However, no differences in PLCγ2 phosphorylation or Syk-phosphorylation were found between RT and 4°C-stored PLTs after stimulation with CVX, and no significant differences in surface expression levels of GPVI were detected between RT and 4°C. Stored platelets in plasma showed superior function after 4°C-storage in aggregation and flow cytometry assays. In contrast, we found similar contractile forces of platelet aggregates when RT-stored or 4°C-stored PLTs were added to platelet-depleted fresh blood. Additionally, at the single cell level, we found a similar magnitude of platelet forces in RT-stored and 4°C-stored PLTs. Similar to human PLTs, mouse PLTs showed significantly more integrin activation, P-selectin exposure, and aggregation in 4°C-stored PLTs compared to RT. To test the recovery of function of stored mouse platelets in vivo, we transfused GFP-positive PLTs into GFP-negative wild-type mice. Contrary to our pre-transfusion results, we found a significantly lower integrin activation response to CVX in 4°C-stored platelets after transfusion, consistent with our previous results in healthy human volunteers. Summary: The in vivo recovery of function of stored PLTs is an underappreciated phenomenon in platelet storage biology, and most studies are solely based on functional in vitro data. Based on our post-transfusion results, storage temperature affects the ability to recover function in vivo significantly in human and mouse platelets. Whether these differences lead to differences in clinical outcomes needs to be investigated in clinical trials. Disclosures Sniadecki: Stasys Medical Corporation: Current equity holder in private company, Other: Co-founder; Curi Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

Description: Erythropoiesis is an intricate process by which lineage-committed erythroid progenitors become mature red blood cells. Reticulocytes are terminal-staged, immature red blood cells with residual RNA after enucleation. In the absence of pathology, reticulocytes are efficiently processed into mature red blood cells and typically represent a small percentage of cells in human peripheral blood. In contrast, when differentiated in vitro from pluripotent stem cells or CD34+ progenitor cells, red cells tend to arrest at the reticulocyte stage. Recent studies have highlighted that uridylation by Terminal Uridylyl Transferases (TUTases) occurs on a broad spectrum of RNA classes in mammalian cells. Oligo-uridylated RNA is recognized by exoribonucleases and targeted for decay. We posited that the machinery behind RNA degradation that accompanies terminal erythropoiesis might involve RNA tail editors coupled to exonuclease activity. Utilizing constitutional murine knockout models, we observed that blood from the TUTase Zcchc6 RNA editor knockout embryos exhibited reticulocytosis and a terminal maturation defect, as documented by FACS, histology, and hematological profiling. Murine strains deficient in the downstream exonuclease Dis3l2 phenocopied the RNA decay defect of the Zcchc6 KO. Conditional knockout murine models of the TUTase-Dis3l2 axis driven by the red cell specific Erythropoietin Receptor-Cre exhibited comparable phenotypes, suggesting a cell intrinsic and niche-independent role for the TUTase-Dis3l2 axis in promoting red blood cell maturation. We are modulating the expression of this axis by various methods to optimize modeling of hemoglobinopathies such as sickle cell anemia. Disclosures No relevant conflicts of interest to declare.

Description: Background: Platelet transfusion is a potentially lifesaving procedure, used for both prophylactic and therapeutic indications. Platelets can be stored at room temperature (RT) for up to 7 days in air-permeable bags. Platelet function diminishes during storage, a phenomenon known as the storage lesion. We and others have shown that platelets can be stored for extended periods of time at 4°C and still show acceptable in vitro function while limiting bacterial growth. In the present study, we used proteomics to examine the changes in human platelets stored at RT and 4°C with a focus on the glycoprotein (GP) Ib-IX-V complex, the key receptor for platelet adhesion at sites of vessel injury. Study Design/Method: Platelet units from healthy donors were stored in 100% plasma with or without agitation (at 22°C or 4°C, respectively) at a concentration of 3x1011/L and sampled on days 0, 3, 7, and 14. Microparticles were detected by flow cytometry as described previously. For proteomic analysis, platelets were washed and digested with trypsin. Tryptic peptides were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano LC-MS/MS). MS/MS spectra were searched against the human protein database using Proteome Discoverer 2.4 software. A student t-test test was used to determine significant differences in analytes amongst the different storage groups. Results/Finding: Under both storage conditions, GPIbα and GPV decreased significantly over storage time. However, comparison of the decline in these proteins to GPIbβ, GPIX, and other membrane proteins indicated that the mechanisms for this decline differ in the two conditions. At RT, the decrease in GPIbα and GPV appears to be largely proteolytic, given that only a minor concomitant decrease in surface level was seen in the protease-insensitive GPIX and a slight increase in GPIbβ. In addition, a comparable decrease in GPIbα level was not observed when a cytoplasmic GPIbα peptide was assayed, suggesting the extracellular portion had been proteolytically removed. In contrast, at 4°C the decline in GPIbα and GPV was accompanied by a modest decrease in GPIX, and only a small decrease in the ratio of extracellular to cytoplasmic to GPIbα peptide. These results suggested that, at 4°C, in addition to proteolysis, which was attenuated as compared to RT storage, another mechanism was responsible for removal of full-length GPIbα and other polypeptides. One such mechanism that could explain this would be loss of membrane from the platelets during 4°C storage. Indeed, we found that extracellular vesicles accumulated in the platelet supernatant during 4°C to a much higher level than at RT storage. Summary: One of the hallmarks of the platelet storage lesion at RT is shedding of surface membrane proteins including GPIbα. Previous studies in stored mouse and human platelets revealed a cleavage mechanism dependent on the metalloproteinase ADAM17. However, whether GPIbα is lost by the same mechanism in cold-stored human platelets was unknown. Our targeted proteomics analysis confirms that proteolysis is a major cause of GPIbα loss at RT, but is a less prominent mechanism at 4°C. However, another mechanism for membrane protein loss is more prominent at the lower temperature: microvesiculation. Thus, these studies provide new insights into the platelet storage lesion and suggest that measures to prevent them will have to be tailored to the dominant mechanism operating at a particular storage temperature. Disclosures No relevant conflicts of interest to declare.

Description: Background: Current blood banking practices dictate that platelets can only be stored at room temperature (RT) for up to seven days because of functional decline and risk of bacterial overgrowth. A lower storage temperature has the potential to slow platelet metabolism and reduce oxidative damage during storage. In the current study, we quantified by mass spectrometry small molecule thiols and disulfides as markers of oxidative stress in platelets stored in plasma at RT or 4°C. Because glutathione (GSH), γGlu-Cys-Gly, is the major thiol in platelets, we also analyzed metabolites (amino acids and dipeptides) involved in GSH synthesis and degradation cycles. Method: Platelets were acquired from five healthy donors and adjusted to 3 × 1011/L in plasma. The platelets from each donor were split into two bags and stored in plasma at RT or 4°C (with or without agitation, respectively) for up to 14 days. Aliquots from each group were taken at days 0, 3, 7, and 14, and N-ethylamaleimide (NEM) was added to block free thiols before storing the samples at -80°C until analysis. Total and reduced forms of GSH and cysteine including protein-bound disulfides were quantified by mass spectrometry as described previously [Fu X. et al. Scientific Reports. 2019; 9(115):1-9]. Paired t-tests were used to evaluate the differences between the groups. Results: GSH is the major intracellular antioxidant that maintains a reducing environment within cells. After 7 days of storage at RT, reduced GSH decreased drastically from 13.0±1.2 pre-storage to 1.7±0.6 µM (p

Description: Introduction Plasmablastic lymphoma (PBL) is a rare, aggressive large cell lymphoma, first described in 1997. PBL is strongly associated with immunodeficient states, such as HIV infection and solid organ transplantation, but up to one third of cases are reported to occur in immunocompetent patients. The pathogenesis of PBL is incompletely understood, though the oncogenic impact of EBV, in particular in the context of dysregulated immune surveillance, together with acquired abnormalities in the MYC pathway appear to play key roles in many cases. Plasma cell markers such as CD138 and CD38 are typically positive, as well as CD30 in a significant subset. Classical B cell markers such as CD20, CD19 and PAX5 are typically absent. The literature on clinical outcomes in PBL is generally limited to small, single-centre case series. Reports describe an aggressive disease of poor prognosis, with median survival of 8 to 15 months, with one series reporting a longer median survival of 32 months. Methods We retrospectively identified patients diagnosed with PBL between 1999 and 2019 from 16 sites across Australia, the United Kingdom and Canada. Patients aged ≥18 years with confirmed tissue diagnosis of PBL at their local treating centre were included. Factors associated with overall survival (OS) were analysed using Cox regression, stratified by site to account for heterogeneity across sites. Risk time for mortality began on the date of diagnosis and ended on the date of death. Patients who were alive, lost to follow-up or transferred to another centre for care, were censored on the date of last follow-up. Risk factors analysed included age, year of diagnosis, HIV status, MYC rearrangement status, CD30 status, lactate dehydrogenase level, disease stage by Lugano consensus criteria, and bone marrow involvement. Results We identified 197 patients with PBL (Table 1). The median age at diagnosis was 55 years (range 18-95) and there was a male predominance (69%). 37% of patients were HIV positive, 56% were HIV negative and 7% were either not tested or had missing results. Other immunosuppressive risk factors included solid organ transplant, allogeneic stem cell transplant (SCT), and immunosuppressive medication. No immunodeficient state was detected in 44%. Fifty per cent of patients were stage IV at diagnosis. Fifty-four per cent were staged using PET/CT. The median follow-up time from diagnosis was 1.36 years, with the longest follow up out to 18.4 years. There were 87 deaths (44%). For patients receiving first-line treatment with curative intent, the rate of complete remission was 57% (103 of 181 patients). Most patients (53%) received CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone)-based chemotherapy as first line, and 27% treatment of higher intensity than CHOP. Rituximab was administered to 20% and 10% were exposed to proteasome inhibitors as part of first line therapy. Five percent of patients underwent autologous SCT in first remission, and a further 5% after first relapse or later. The median survival time was 4.8 years, with a 5-year OS of 49% and 10-year OS of 45% (figure 1). In multivariate analysis the only adverse factors associated with OS were bone marrow involvement and stage IV disease. Patients without bone marrow involvement at diagnosis had improved OS, compared to those who did (hazard ratio (HR) 0.36, 95%CI 0.18-0.72, p=0.004) (figure 2). There was an increasing trend for mortality with higher disease stages (p-trend=0.002). The median survival was 14.1 years for stage I, 10.7 years for stage II, 5.1 years for stage III and 1.2 years for stage IV. However, only stage IV disease was independently associated with inferior OS in multivariate analysis (HR 2.93, 95%CI 1.43-6.00, p=0.003) (figure 3). OS did not change depending upon year of diagnosis. Conclusion We report a multinational retrospective cohort of patients diagnosed with PBL and to our knowledge the largest single series of PBL to date. OS was longer than previously published data, particularly in patients with early-stage disease. However, patients with stage IV disease and baseline bone marrow involvement had inferior OS. HIV infection did not affect outcome. These findings suggest that baseline bone marrow biopsy and PET staging are useful prognostic tools. There is also an ongoing need for the evaluation of the predictive value of PET imaging and novel agents in PBL, especially in higher-risk disease. Disclosures Di Ciaccio: Jansen: Honoraria, Other: travel and accomodation grant. Cwynarski:Takeda: Consultancy, Other: Conference/travel support; Roche: Consultancy, Other: Conference/travel support. Burton:Celgene: Honoraria; Leeds Teaching Hospitals NHS Trust: Current Employment; Takeda: Honoraria, Other: Travel Support; BMS: Honoraria; Roche: Honoraria, Other: Travel Support. Kuruvilla:Antengene: Honoraria; Janssen: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy; AstraZeneca Pharmaceuticals LP: Honoraria, Research Funding; Merck: Consultancy, Honoraria; Celgene Corporation: Honoraria; Amgen: Honoraria; TG Therapeutics: Honoraria; Pfizer: Honoraria; Novartis: Honoraria; Bristol-Myers Squibb Company: Consultancy. McKay:Greater Glasgow and Clyde Health Board: Current Employment; Roche, Gilead, Takeda, Janssen: Other: For lectures etc; Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BeiGene: Membership on an entity's Board of Directors or advisory committees; Janssen: Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Speakers Bureau; TAKEDA: Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Speakers Bureau. Linton:BeiGene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Conference/travel support; Roche: Consultancy, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Patents & Royalties; Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Hartley-Taylor: Honoraria; The Christie NHS Foundation Trust and The University of Manchester: Current Employment. Manos:Bristol-Myers Squibb: Other: Conference sponsorship. Hamad:Abbvie: Honoraria; Novartis: Honoraria.